Exosome transfer of nucleic acids to cells

ABSTRACT

Methods for introducing nucleic acids to cells via exosomes for use in gene modulation and therapy, such as for gene silencing and to introduce genetic material into cells to compensate for abnormal genes or to induce or repress a process in the recipient cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application Ser.No. 60/797,149, filed May 3, 2006, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is based on the unexpected findings that exosomesreleased to the extracellular milieu can carry selective RNA from theparental cells. This can, according to this invention, be used totransfer genetic material to recipient cells by exosomes. Bytransferring nucleic acids to recipient cells, exosomes affect anothercell's (recipient cells) protein machinery and thus protein content, andthe invention demonstrates for the first time that nucleic acids, forexample RNA and DNA, deliberately can be transferred between the cellsor organs using exosomes, and can thus be utilized for gene modulationand therapy in mammalian cells.

2. Description of the Related Art

Exosomes are small membrane vesicles of endocytic origin that arereleased into the extracellular environment following fusion ofmultivesicular bodies with the plasma membrane. The size of exosomesranges between 30 and 100 nm in diameter. Their surface consists of alipid bilayer from the donor cell's cell membrane, and they containcytosol from the cell that produced the exosome, and exhibit membraneproteins from the parental cell on the surface.

Exosomes exhibit different composition and function depending on thecell type from which they are derived. There are no “exosome-specific”proteins; however several proteins identified in these vesicles areassociated with endosomes and lysosomes reflecting their origin. Mostexosomes are enriched in MHC I and II (major histocompatibility complexI and II; important for antigen presentation to immunocompetent cellssuch as T-lymphocytes), tetraspanins, several heat shock proteins,cytoskelatal components such as actins and tubulins, proteins involvedin intracellular membrane fusion, signal transduction proteins andcytosolic enzymes.

Exosomes are produced by many cells including epithelial cells, B and Tlymphocytes, mast cells (MC) as well as dendritic cells (DC). In humans,exosomes have been found in blood plasma, urine, bronchoalveolar lavagefluid, intestinal epithelial cells and tumor tissues.

All functions of exosomes have not been elucidated, but data stronglyindicates they mediate communication between cells. This communicationcould take place in different ways. First, exosomes could bind to cellsurface receptor in a similar way as cell to cell interaction. Second,exosomes could attach to the cell membrane and give the cells newreceptors and properties. Thus, exosomes can also fuse with target cellsand exchange membrane proteins and cytosol between two cell types.

We have put extra effort into understanding the content and biologicalfunction of exosomes specifically released by mast cells. In proteomicassays we have found that these exosomes contain a larger number ofproteins than previously understood. However, the unique finding fromour research is that we have discovered a substantial amount ofselective RNA in exosomes from mast cells. Furthermore, use of differentrecipient cells displays an uptake of exosomal RNA indicating transferof genetic material from exosomes into recipient cells, which in turnwill lead to translation of specific protein in the target cells.

Considering the exosomal protein content and their capacity tocommunicate with different recipient cells, it is particularly useful tobe able to modify the genetic content of exosomes in order to add orregulate a gene in recipient cells. The method using the exosomes'capacity of carrying specific genetic material and transferring it torecipient cells is described in this application. In this method, therecipient is affected in its function, as well as in its ability to stayalive, further develop, proliferate or mature.

The method is unique and different from any previous described methods.Several patents and patent applications use exosomes to influence theimmune system through stimulatory or inhibitory function via exosomalprotein interaction with immune cells, and for treatment of viraldisease by influencing the immune system. It has been suggested thatexosomal proteins can be modified by mutation to affect the immunesystem. However, no patent or patent application or any publiclyavailable information that we have found describes or suggests use ofexosomes to transfer genetic material or nucleic acids to cells.

SUMMARY OF THE INVENTION

The present invention discloses novel methods of delivering nucleic acidconstructs by exosomes to recipient cells. The invention method includesRNA and DNA constructs that are transferred into exosomes by using theirparental cells (transformation, transfection, or modification of theparental cells), or using conventional methods for instance,transformation and transfection, to introduce nucleic acids directlyinto exosomes. The method of the invention is an excellent tool for genetherapy to introduce genetic material into recipient cells to compensatefor abnormal genes or to introduce RNA or DNA that produces proteinsthat affect the function of the recipient cells or their ability to stayalive or develop or mature. For gene therapy, usually viruses orliposomes are used as vectors for transfer of genetic material, becausethey can deliver the new gene by infecting the cell. Use of exosomes hasseveral advantages compared to conventional methods, and mostimportantly exosomes are derived from cells, and possibly even therecipient's own cells, and are thus not a foreign body for the immunesystem, avoiding adverse immune reactions. Exosomes are thus easilyproduced, isolated and modified for use in gene modulation and therapy.

Other objects and features of the inventions will be more fully apparentfrom the following disclosure and appended claims.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

Using exosome vesicles for transferring genetic material, nucleic acids,to the cytosol or nucleus of a cell as described in the inventionherein, can treat inherited diseases, cellular or body dysfunctions,induce or repress cell death (apoptosis), change cellular ageing, inducetolerance, re-direct existing immune responses, change intracellularactivity or cellular behaviour. It can also be used in all kinds of genetherapy of genetic disorders, malignant diseases or diseases involvingimmune cells or any other cell type in the body including vasculature,epithelial cells, interstitial cells, musculature, skeletal system,nervous system, liver cells, kidney cells, gut cells, lung cells, skincells or any other cell in the body.

Genetic material that can be transferred by exosomes, described in theinvention herein is: microRNA, mRNA, tRNA, rRNA, siRNA, regulating RNA,non-coding and coding RNA, DNA fragments, and DNA plasmids, includingnucleic acids of any type. The methods of transferring the geneticmaterial (constructs of DNA or RNA, or any type of nucleic acids)directly into exosomes are transformation, transfection andmicroinjection.

Genetically dissimilar exosomes can be isolated for further delivery torecipient cells using either their donor cells or by introducingspecific nucleic acids into them. Different approaches for production ofexosomes containing a set of genetic material are described below.

Different procedures for production of genetic dissimilar exosomes usingthe exosome producing cells for further delivery to recipient cellsinclude:

(a) Exosomes from different donor cells. Exosomes containing differentset of genetic material can be isolated from different donor cells (e.g.B and T lymphocytes, mast cells, and dendritic cells) for furtherdelivery to recipient cells. Since the genotype of the donor cells aredissimilar, which is due to their role and environment, the isolatedexosomes from different cell type give rise to exosomes with dissimilargenotypes.

(b) Exosomes from different conditions. Growth of the exosome producingcells in a certain condition give rise to cellular changes, andsubsequently to genetically modified exosomes. The exosomes cansubsequently be used for transferring the set of nucleic acids torecipient cells.

(c) Exosomes from different human being or disease. Exosomes fromdifferent persons or diseases contain diverse set of nucleic acids. Thegenotype of the exosomes is different due to their donor cell's sourceand conditions. The exosomes with the unique set of nucleic acids can beisolated for further delivery of the nucleic acids to recipient cells.

(d) Exosomes from genetically modified donor cells. Gene disruption ormutations in exosome producing cells give rise to genetically modifiedexosomes that can be used for delivery of their nucleic acids torecipient cells. Expression of any kind of nucleic acids present inexosomes can be influenced (erased or up/down regulated) by genemanipulation of the exosome producing cells. For instance, disruption ofa gene in exosome producing cells results in a lack of correspondingmRNA in exosomes. Simultaneously, up or down-regulation of a gene inexosome producing cells affects amount of corresponding mRNA inexosomes.

Specific nucleic acid construct(s) (cloned genes, DNA fragments andplasmids, microRNA, siRNA, coding and none coding DNA and RNA) can betransferred to recipient cells via genetic modified exosomes. Thenucleic constructs can be manufactured using standard techniques e.g.cloning, isolation and amplification of RNA or DNA sequences, forfurther transformation into exosomes.

(a) Insertion of specific constructs into exosomes using their donorcells. New constructs (RNA and DNA) can be introduced into exosomesusing their donor cells. The nucleic acid constructs can be introducedinto the donor cells (the exosome producing cells) and consequently bythe intracellular functions the transcript or the constructed by itselfwill be translocated into exosomes.

(b) Insertion of specific constructs directly into exosomes. Exosomescan be isolated from different origin, e.g. in vitro growing cells,human body, or cells originated from a human and animal. Geneticconstructs of RNA or DNA can be introduced into these exosomes directlyby using conventional molecular biology techniques such as in vitrotransformation, transfection, and microinjection . The geneticallymodified exosomes can further be used for transferring their nucleicacids to recipient cells.

Methods for Production of Genetically Modified Exosomes

Transformation or transfection of genetic material into exosomes Moregenerally the term transformation and transfection is used to describemechanisms of DNA and RNA transfer in molecular biology and it wasdescribed for the first time 1944 by Oswald Avery, Colin MacLeod, andMaclyn McCarty (Lederberg J., Genetics. 1994 February;136(2):423-6).

(a) Electroporation. By this methods number of holes is made incells/exosomes by briefly shocking them with and electric field of100-200 V/cm. The DNA/RNA can enter the cells/exosomes through the holesthat made by the electric field.

(b) Lipofection. The method commonly called transfection and can be usedto transform cells/exosomes with DNA/RNA via vesicles containing thedesired genetic constructs. The vesicles fuses with the cell membrane(similar to how two oil spots at the top of a broth will fuse) and thecontents of the vesicles and the cells are combined. There are a numberof transfection kits in the market, ready for use, e.g. DeliverX siRNATransfection Kit (cat. No. DX0002) from Panomics, FuGENE® HDTransfection Reagent (Cat. no. 04709691001) from Roche andLIPOFECTAMINE™ 2000 (Cat. No. 11668-027) from Invitrogen.

(c) Transformation using heat shock. Chilling cells/exosomes in thepresence of divalent cations such as Ca²⁺ (in CaCl₂) makes theirmembranes become permeable to RNA or DNA plasmids/fragments fragments.Cells/exosomes are incubated with the DNA and then briefly heat shocked(42° C. for 30-120 seconds), which causes the DNA to enter the cell.This method works well for circular plasmid DNAs.

The above methods describe briefly how genetically modified exosomes canbe achieved to transfer RNA and DNA to recipient cells. Exosomes thatcontain RNA/DNA or that are modified to contain the gene of interestwill be isolated and shifted to the recipient cells, to affect theirbiological function or survival. Consequently, the exosomes will disposetheir content into the cytoplasm of the target cells, which in turnleads to translation of mRNA to specific proteins in the target cellthrough the cells own protein machinery. Further exosomes are capable tocarry and transfer small coding and none coding RNA such as microRNA andsiRNA that may regulate translation of a specific gene.

Exosomes being vesicles as carrier of DNA or RNA as described in theinvention herein can be used to treat inherited diseases inhematopoietic, non-hematopoietic, stem cells, and organs. Exosomevesicles can also be used as carriers of DNA or RNA constructs fortreatments of microbiological infections or diseases or dysfunctions inhumans or animals, or transfer of genetic material of any biologicalmembrane.

Because in humans CD4 T-cells are the target for HIV infections,infected cells can be treated with RNA or DNA constructs (siRNA, RNAi,or DNA) carried and transferred by exosomes to the infected T-cells,specifically designed for silencing of the translation of the viral RNA.Thus our invention discloses that exosomes that are capable oftransferring their nucleic acids to CD4 T-cells can be used fortreatment of HIV infected T-cells, as well as T-cell malignancies suchas lymphoma or lymphatic leukemias.

Changing or modifying the genetic material of exosomes by altering thecondition for the exosome-producing cells, is achieved by changing pH,temperature, growing conditions, or using antibodies/chemicals towardexosome-producing cells. This results in alteration of the nucleic acidcontent. Also, over-expression or repression of cytokines, chemokinesand other genes in the exosome-producing cells can be used to change ormodify the genetic content of exosomes

Transferring sense or anti-sense RNA to specific cells using exosomevesicles to switch off genes instead of adding new ones results in downregulation (slow down) or prevention of translation of the particulargene. The method is called RNA interference (siRNA).

The invention herein makes it possible to transfer genetic materials invitro to stem-cells acquired from a patient or a donor prior toadministration of this stem cell to a patient or a recipient human, oranimal.

To administer nucleic acids to recipient cells or tissues, DNA orRNA-containing exosomes can be administered to cells by addition of theexosomes to cell cultures in vitro, or injection of these exosomesintravenously, or by any other route, in vivo as is known in the art.Exosomes can be targeted to any cell in the body, including cells in thecardiovascular system, skeletal muscle cells, joint cells, neural cells,gut cells, lung cells, liver cells or kidney cells, or cells in theimmune system, or to any type of cell with any function or dysfunctionin the body of humans or animals, including malignant cells.

As disclosed in the invention herein, exosomes can be used to delivergenetic material to recipient cells to use the cell's own proteinmachinery to produce any drug or precursor of any drug, or to affect thefunction or metabolism of any drug, in any cell in humans or animals.

To avoid interference with undesirable or irrelevant genetic material,it is preferable to use exosomes that are lacking genetic contents.Empty exosomes can be used for direct transfer to recipient cells or fordirect transfection/transformation of a specific gene (RNA or DNA) intoexosomes.

Detection of Mast Cell Derived Exosomes.

Exosomes released from the murine mast cell line MC/9 (ATCC Manassas,Va., USA, Number: CRL-8306) were isolated, adsorbed to carbon-coatedgrids, and detected using electron microscopy. For detection of theexosome specific surface protein CD63, exosomes were also purified fromboth primary bone marrow mast cells (BMMC) and the cell line MC/9. Theexosomes were adhered onto aldehyde beads and stained with a CD63antibody followed by a secondary PE-coupled antibody. These beads wereanalysed using flow cytometric detection, confirming the presence ofCD63 on the surface of both BMMC and MC9 derived exosomes.

Identification of Exosomal Proteins.

To understand the biological function of the mast cell derived exosomes,the protein content was analysed using nano-flow LC-MS/MS. The totalprotein content was extracted from the isolated exosomes and collectedon a SDS-PAGE gel. The protein band was trypsinated and analysed byLC-MS/MS. The results of all the tandem mass spectra were searched bythe MASCOT (Matrix Science, London) program for identification. Theresults revealed that these exosomes contain a larger number of proteinsthan were previously known. Approximately 150 proteins were identified,many of which are associated with cellular transcription, translation,and protein folding. The identified proteins include several ribosomalproteins as well as heat shock proteins, chaperones, annexines,cytoskeleton proteins and membrane-bound proteins such as CD63, CD54,CD43 and MHC class I.

Detection of DNA and RNA from Mast Cell Derived Exosomes.

Since a certain number of the identified proteins are associated withRNA and the transcriptional machinery, we hypothesised that exosomes mayalso contain DNA and/or RNA. To examine this, we performed RNA and DNAextraction from both exosomes and the full exosome producing mast cells.Presence of DNA and RNA were determined by spectrophotometry and on anagarose gel. No DNA could be detected in the exosome sample, whereas asubstantial amount of selective RNA was observed from the exosomes.Specifically exosomal RNA contained very low, or undetectable, amountsof ribosomal RNA, which indicates the presence of other types of RNA,i.e. mRNA.

Microarray Analysis of Exosomal RNA.

In order to characterize the RNA from the mast cell derived exosomes,Affymetrix mouse DNA microarray (Affymetrix) was applied using RNA fromboth mast cells and their exosomes. The results revealed that exosomescarry mRNA from approximately 2500 genes, which is approximately 10% ofgenes that are expressed in the mother mast cells. Furthermore, the geneprofile analysis displayed essential differences in the mRNA between theexosomes and their parental cells. The most abundant transcripts in theexosomes differ from abundant transcripts in the parental cells whichindicate selectivity of the exosomal RNA. Interestingly, the exosomescarried mRNA from 180 genes whose transcript were absent in their mothermast cells. The results indicate that mRNA is transported into exosomesin a highly selective way and it seems that the parental cells express acertain number of genes exclusively for exosomal delivery.

Detection of RNA from BMMC Exosomes.

To identify RNA in exosomes from a non-cell line source, bone marrowcells were harvested from mice and cultured to become mast cells (BoneMarrow derived Mast Cells: BMMC) for 4 weeks. During the last 48 hours,the cells were cultured in the presence of radioactive uracil( seeexample 1). The exosomes from the culture were isolated and the RNAlabelled with radioactivity was detected by scintillator counting. Theresults showed that the incorporated radioactive uracil in cellular RNAcould be found in exosomes. The amount of RNA from the bone marrowderived exosomes is not as abundant as in the mast cell line exosomes,which can be explained by a lower number of BMMC cells used for theharvest, and the in vitro growth condition of these cells.

Transfer of RNA by Exosomes Between Cells.

In order to examine whether exosomes can transfer the mRNA they containto another cell, mast cell derived exosomes containing radioactiveuracil mRNA were added to cultured dendritic cells (DC), CD4 T-cells,and MC/9 mast cells. Samples from the cultures were taken at differentintervals and the cells were isolated and washed by centrifugation. RNAfrom the recipient cells was isolated and examined for radioactiveuracil using a scintillator. Most importantly, DC, CD4 and MC/9 cellsexposed to the exosomes contained increased amount of radioactiveuracil, thus having been absorbed from the exosomes. The results showthat mast cell derived exosomes can transfer RNA to other cells, in thiscase DC, CD4 and MC/9 cells.

The data show that mRNA can be transferred between two mammalian cellsthrough exosomes. Biologically, this suggests that one cell can affectanother cell's protein production by signalling via exosomes. This hassubstantial biotechnological applications, since exosomes may be used ascarriers to deliver mRNA, or DNA-probes, to target cells such asmalignant cells or cells in the immune system. Therefore according tothis invention, the exosomes provide a vehicle for gene modulation andtherapy that is likely to be without the side effects of other genetherapy vehicles, such as viruses or other types of lipid bodies.

The features of the present invention will be more clearly understood byreference to the following examples, which are not to be construed aslimiting the invention.

EXAMPLE 1

Cell Preparation

MC/9 cells (ATCC) were cultured according to manufacturer'srecommendations. To eliminate exosomes present in serum, Rat T-Stim andFBS were ultracentrifuged at 120 000 g for 90 min using a Ti70 rotor(Beckman optima LE-80k Ultracentrifuge). The human mast cell line HMC-1(Dr Joseph Butterfield, Mayo Clinic, USA), was cultured in IMDMcontaining 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine and 1.2 mM alph-thioglycerol. For release of exosome, theHMC-1 cells were cultured in the presence of 1 μM calcium ionophore for30 min. Bone marrow mast cells (BMMC) were prepared by culturing bonemarrow cells from femurs of 7-10 wk old male BALB/c in the presence ofIL-3 (R&D systems) as described previously (Razin, E. et al. Interleukin3: A differentiation and growth factor for the mouse mast cell thatcontains chondroitin sulfate E proteoglycan. J Immunol. 132, 1479-1486(1984)). After 4 weeks of culture, the cells were harvested andconsisted of 96% pure MCs as analysed by morphology. During the last 48h, BMMC were cultured at 3×10⁶ cells/ml in complete medium withultracentrifuged FBS supplemented with 10 ng/ml IL-4 (R&D-systems), andin some experiments in the presence of 1 μl/ml ³H-Uracil (AmershamBiosciences). For culture of CD4⁺ T cells, mouse spleens were collectedand passed through a 70 μm followed by 30 μm filter. CD4⁺ T cells werepurified by negative selection using the SPINCEP® mouse CD4⁺ T cellsenrichment cocktail (Stemcell Technologies) according to themanufacturer's instructions. The purity of the CD4⁺ T cells ranged from89 to 91%, as analysed by flow cytometry. The cells were cultured inRPMI 1640 containing 10% FBS, 100 U/ml penicillin and 100 μg/mlstreptomycin at 1×10⁶ cells per ml in flat bottom 48 well plates.

EXAMPLE 2

Exosome Purification

Exosomes were prepared from the supernatant of MC/9, BMMC and HMC-1cells by differential centrifugations as previously described (Raposo,G. et al. B lymphocytes secrete antigen-presenting vesicles. J. Exp.Med. 183, 1161-1172 (1996)) . Cells were harvested, centrifuged at 500 gfor 10 min to eliminate cells and at 16,500 g for 20 min, followed byfiltration through 0.22 μm filter to remove cell debris. Exosomes werepelleted by ultracentrifugation (Beckman Ti70 rotor) at 120 000 g for 70min. For mass spectrometry, the exosome pellet was washed once in PBS.Exosomes were measured for their protein content using BCA™ ProteinAssay Kit (Pierce). For the density gradient experiment the 120,000 gexosome pellet was floated in a sucrose gradient (0.25-2 M sucrose, 20mM Hepes/NaOH, pH 7.2). The exosomes were dissolved in 2.5 M sucrose andthe gradient was layered on top of the exosome suspension. The gradientwas centrifuged at 100,000 g for 15 h according to (Rapso et al 1996).Briefly, the gradient fractions (10×3.8 ml) were collected from thebottom of the tube, diluted with 10 ml PBS and ultracentrifuged for 2 hat 150 000 g (Beckman Ti70.1 rotor), and the pellets were extracted byTrizol® (Invitrogen).

EXAMPLE 3

Isolation of RNA, DNA and Proteins

RNA, DNA and proteins were isolated using Trizol® (Invitrogen) orRNEASY® mini kit (Qiagen) according to the manufacturer's protocol. Forco-purification of microRNA and total RNA, the RNA was extracted usingTrizol, followed by the RNEASY® mini kit. Cells and exosomes weredisrupted and homogenized in Buffer RLT (Qiagen) and 3.5 volumes of 100%ethanol were added to the samples prior use of the RNEASY mini spincolumn. The rest of the procedure was performed according to themanufacturer's protocol.

EXAMPLE 4

Introducing DNA/RNA Fragments or Constructs into Exosomes

Insertion of Specific Constructs Directly into Exosomes.

Exosomes can be isolated from different origin, e.g. in vitro growingcells, human body, or cells originated from a human and animal. Geneticconstructs of RNA or DNA can be introduced into these exosomes directlyby using conventional molecular biology techniques such as in vitrotransformation, transfection, and microinjection.

EXAMPLE 5

Administration of DNA or RNA-Containing Exosomes to Cells

Transfer Experiments

To label MC/9 exosome RNA, cells were cultured in complete mediumsupplemented with 1 μl/ml ³H-Uracil 72 h before exosome isolation.Exosomes were isolated according to the isolation protocol and washed byultra-filtration (10 kDa, Millipore) to remove free nucleotides. Theexosomes were added to MC/9, CD4⁺, and HMC-1 cells in the ratio of 8:1between donor cells and recipients at the starting point of labeling. At0 h and 24 h, cells were harvested and washed twice. RNA was isolated byRNEASY® mini kit and the signal of radioactive RNA was measured usingscintillation. Medium supplemented with 1 μl/ml ³H-Uracil absent fromdonor cells was treated equally and used as negative control.

In vitro Translation

Total exosomal RNA was purified using RNEASY® mini kit and 0.5 μg wasused for the translation. The in vitro rabbit lysate translation kit(Promega Corporation) was used according to the manufacturer'srecommendation to translate exosomal mRNA to proteins. A sample withoutexosomal RNA was treated equally and used as negative control. After thetranslation procedure was accomplished, total proteins were precipitatedusing acetone and determined using RC DC protein assay (BioRad). Theprotein content of the samples (presence and absence of the exosomalRNA) was compared using 2D-PAGE, BioRad instruments (Mini-protean® 3cell) and recommendation. The 2D-gels were visualized using SyproRuby(BioRad) and digitalized using phosphoimager. Protein spots of thesamples were compared and a selection of the newly produced proteins wascut, trypsinated, and identified using LC-MS/MS followed by MASCOTprogram search. The newly produced proteins of mouse origin werecompared to the genes identified from the DNA microarray analysis.

In Vivo Translation

MC/9 exosomes (1000 μg) were added to HMC-1 cells (8×10) in threedifferent time points (0, 3, 6 h) and the cells were incubated forapproximately 24 h. The HMC-1 cells were harvested, washed, and thetotal proteins of the cells were separated by 2D-PAGE according toProteomics Core facility. A sample without exosomes was treated equallyand used as negative control. The newly produced proteins were detectedusing PDQUEST and 96 spots were cut and identified using MALDI-toffollowed by MASCOT program search, according to Proteomics Core Facility(University of Gothenburg).

The exosomes then deliver the nucleic acids to recipient cells andconsequently affect their biological function or survival.

EXAMPLE 6

Production of Exosomes that are Lacking Genetic Material

The empty exosomes are used for direct transfer to recipient cells orfor direct transfection/transformation of a specific gene (RNA or DNA)into exosomes. The methods to produce empty exosomes (empty of geneticmaterial) are multiple as known by one skilled in the art; includingUV-exposure, mutation of proteins that carry RNA into exosomes, as wellas electroporation and chemical treatments to open pores in the exosomalmembranes. The methods include mutation/deletion of any protein that canmodify loading of any nucleic acid into exosomes.

EXAMPLE 7

Production of Mouse Proteins in Human Mast Cells after Transfer of MouseMC/9 Exosomes

To test whether mouse proteins could be produced in human mast cellsafter transfer of mouse MC/9 exosomes, we determined the presence ofmouse proteins in the recipient cell, by 2D-PAGE followed by MALDI-tof.After incubation of the human cells with mouse MC/9 exosomes for 24hours, 96 new or enhanced protein spots were identified. Interestingly,three distinct mouse proteins were identified in the human cells thatare not present in MC/9 exosomes. These proteins were mouse CDC6(089033), mouse Zinc finger protein 271 (P15620) and mouse CX7A2(P48771). The mRNA of the first two proteins was present in two of themicroarray experiments, and the last one was present in all fourmicroarrays performed, suggesting that mRNA delivered by exosomes to arecipient cell can be translated to proteins.

The proteomic results from transfer of MC/9 exosomes to HMC-1 cells whenhuman mast cells HMC-1 were incubated with the mouse MC/9 exosomes andwithout for 24 hours show that mouse proteins could be produced in thehuman mast cells. Proteins between the two gels were matched and 96newly produced proteins were identified by MALDI-tof and mouse proteinswere produced from the exosomal mRNA.

EXAMPLE 8

Using Exosomes as Gene Therapy of Malignant Disease

Exosomes are produced by malignant cells, taken from a patient sufferingfrom a malignant disease. These exosomes are processed to containgenetic constructs of any type or specificity, to be reintroduced to thepatient. The exosomes from the malignant cell then preferentially fusewith cells of the same type, which deliver the DNA and or RNA constructsto the malignant cell specifically, as gene therapy of malignantdisease. The procedure is performed according to example 1-6, but withexosomes produced by malignant cells.

While the invention has been described with reference to specificembodiments, it will be appreciated that numerous variations,modifications, and embodiments are possible, and accordingly, all suchvariations, modifications, and embodiments are to be regarded as beingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method of transferring genetic material,comprising in this order: a) isolating exosomes produced and released bya donor cell; b) modifying the isolated exosomes to contain selectednucleic acid constructs; and c) administering the exosomes containingthe selected nucleic acid constructs to recipient cells, whereinadministering the exosomes results in the transfer of the specificnucleic acid constructs from the exosomes to the cytoplasm of therecipient cells; wherein the isolated exosomes do not contain anysubstantial amount of nucleic acids; and further wherein the selectednucleic acid constructs are added to the exosomes that do not containany substantial amount of nucleic acids.
 2. The method of claim 1,wherein the selected nucleic acid constructs are selected from the groupconsisting of mRNA, tRNA, rRNA, siRNA, microRNA, regulating RNA,non-coding and coding RNA, DNA fragments, and DNA plasmids.
 3. Themethod of claim 1, wherein the recipient cells are selected from thegroup consisting of cells from recipients having inherited diseases inhematopoietic, non-hematopoietic, stem cells, and organs.
 4. The methodof claim 1, wherein the recipient cells comprise CD4T-cells fortreatment of HIV infected T-cells.
 5. The method of claim 1, wherein theselected nucleic acid constructs are transferred to treat diseases,induce or repress cell death, change cellular ageing, induce tolerance,re-direct existing immune responses, or change intracellular activity orcellular behavior.
 6. The method of claim 1, wherein the exosomes areisolated by a method selected from the group consisting of isolationfrom a specific donor cell, isolation from a person with a particulardisease or condition; and isolation from a genetically modified donorcell.
 7. The method of claim 6, further comprising genetically modifyingthe donor cell to erase production of a nucleic acid, or to up-regulateor down-regulate production of a nucleic acid.
 8. The method of claim 1,further comprising introducing the selected nucleic acid constructs intothe exosomes using a method selected from the group consisting oftransformation, transfection and microinjection of the selected geneticmaterial directly into exosomes.
 9. The method of claim 1, wherein theselected nucleic acid constructs are administered to the recipient cellsby a method selected from the group consisting of addition of theexosomes to cell cultures in vitro, intravenous injection of theexosomes, in vivo administration, and administration targeted toparticular cells in the body.
 10. The method of claim 1, wherein theexosomes are isolated from a patient with a malignant disease; and arefurther processed to contain selected nucleic acid constructs, and thenare reintroduced into the patient.
 11. The method of claim 1, whereinthe exosomes are obtained from tumor tissue.
 12. The method of claim 1,wherein the exosomes are obtained from intestinal epithelial cells,lymphocytes, mast cells or dendritic cells.
 13. The method of claim 1,wherein the exosomes are obtained from in vitro growing cells.
 14. Themethod of claim 13, wherein the exosomes are isolated from cell culturesupernatant.
 15. The method of claim 1, wherein the exosomes areisolated from body fluid.
 16. The method of claim 15, wherein the bodyfluid comprises blood plasma, urine or bronchoalveolar fluid.